Current knowledge and recent advances in understanding metabolism of the model cyanobacterium Synechocystis sp. PCC 6803

Cyanobacteria are key organisms in the global ecosystem, useful models for studying metabolic and physiological processes conserved in photosynthetic organisms, and potential renewable platforms for production of chemicals. Characterising cyanobacterial metabolism and physiology is key to understanding their role in the environment and unlocking their potential for biotechnology applications. Many aspects of cyanobacterial biology differ from heterotrophic bacteria. For example, most cyanobacteria incorporate a series of internal thylakoid membranes where both oxygenic photosynthesis and respiration occur, while CO2 fixation takes place in specialised compartments termed carboxysomes. In this review, we provide a comprehensive summary of our knowledge on cyanobacterial physiology and the pathways in Synechocystis sp. PCC 6803 (Synechocystis) involved in biosynthesis of sugar-based metabolites, amino acids, nucleotides, lipids, cofactors, vitamins, isoprenoids, pigments and cell wall components, in addition to the proteins involved in metabolite transport. While some pathways are conserved between model cyanobacteria, such as Synechocystis, and model heterotrophic bacteria like Escherichia coli, many enzymes and/or pathways involved in the biosynthesis of key metabolites in cyanobacteria have not been completely characterised. These include pathways required for biosynthesis of chorismate and membrane lipids, nucleotides, several amino acids, vitamins and cofactors, and isoprenoids such as plastoquinone, carotenoids, and tocopherols. Moreover, our understanding of photorespiration, lipopolysaccharide assembly and transport, and degradation of lipids, sucrose, most vitamins and amino acids, and heme, is incomplete. We discuss tools that may aid characterisation of cyanobacterial metabolism, notably CyanoSource, a barcoded library of targeted Synechocystis mutants, which will significantly accelerate characterisation of individual proteins

Emerging Species and Genome Editing Tools: Future Prospects in Cyanobacterial Synthetic Biology

Recent advances in synthetic biology and an emerging algal biotechnology market have spurred a prolific increase in the availability of molecular tools for cyanobacterial research. Nevertheless, work to date has focused primarily on only a small subset of model species, which arguably limits fundamental discovery and applied research towards wider commercialisation. Here, we review the requirements for uptake of new strains, including several recently characterised fast-growing species and promising non-model species. Furthermore, we discuss the potential applications of new techniques available for transformation, genetic engineering and regulation, including an up-to-date appraisal of current Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein (CRISPR/Cas) and CRISPR interference (CRISPRi) research in cyanobacteria. We also provide an overview of several exciting molecular tools that could be ported to cyanobacteria for more advanced metabolic engineering approaches (e.g., genetic circuit design). Lastly, we introduce a forthcoming mutant library for the model species Synechocystis sp. PCC 6803 that promises to provide a further powerful resource for the cyanobacterial research community

The quantum metrology triangle and the re-definition of the SI ampere and kilogram; Analysis of a reduced set of observational equations

We have developed a set of seven observational equations that include all of the physics necessary to relate the most important of the fundamental constants to the definitions of the SI kilogram and ampere. We have used these to determine the influence of alternative definitions being considered for the SI kilogram and ampere on the uncertainty of three of the fundamental constants (h, e and mu). We have also reviewed the experimental evidence for the exactness of the quantum metrology triangle resulting from experiments combining the quantum Hall effect, the Josephson effects and single-electron tunnelling.Comment: 16 pages, 3 figures & 5 table

Fetal heart rate responses in chronic hypoxaemia with superimposed repeated hypoxaemia consistent with early labour: a controlled study in fetal sheep

Objective: Deceleration area (DA) and capacity (DC) of the fetal heart rate can help predict risk of intrapartum fetal compromise. However, their predictive value in higher risk pregnancies is unclear. We investigated whether they can predict the onset of hypotension during brief hypoxaemia repeated at a rate consistent with early labour in fetal sheep with pre-existing hypoxaemia. Design: Prospective, controlled study. Setting: Laboratory. Sample: Chronically instrumented, unanaesthetised near-term fetal sheep. Methods: One-minute complete umbilical cord occlusions (UCOs) were performed every 5 minutes in fetal sheep with baseline paO2 17 mmHg (normoxic, n = 11) for 4 hours or until arterial pressure fell <20 mmHg. Main outcome measures: DA, DC and arterial pressure. Results: Normoxic fetuses showed effective cardiovascular adaptation without hypotension and mild acidaemia (lowest arterial pressure 40.7 ± 2.8 mmHg, pH 7.35 ± 0.03). Hypoxaemic fetuses developed hypotension (lowest arterial pressure 20.8 ± 1.9 mmHg, P P = 0.04) and final (P = 0.012) 20 minutes of UCOs. DA was not different between groups. Conclusion: Chronically hypoxaemic fetuses had early onset of cardiovascular compromise during labour-like brief repeated UCOs. DA was unable to identify developing hypotension in this setting, while DC only showed modest differences between groups. These findings highlight that DA and DC thresholds need to be adjusted for antenatal risk factors, potentially limiting their clinical utility

A toolbox to engineer the highly productive cyanobacterium Synechococcus sp. PCC 11901

Synechococcus sp. PCC 11901 (PCC 11901) is a fast-growing marine cyanobacterial strain that has a capacity for sustained biomass accumulation to very high cell densities, comparable to that achieved by commercially relevant heterotrophic organisms. However, genetic tools to engineer PCC 11901 for biotechnology applications are limited. Here we describe a suite of tools based on the CyanoGate MoClo system to unlock the engineering potential of PCC 11901. First, we characterised neutral sites suitable for stable genomic integration that do not affect growth even at high cell densities. Second, we tested a suite of constitutive promoters, terminators, and inducible promoters including a 2, 4-diacetylphloroglucinol (DAPG)-inducible PhlF repressor system, which has not previously been demonstrated in cyanobacteria and showed tight regulation and a 228-fold dynamic range of induction. Lastly, we developed a DAPG-inducible dCas9-based CRISPR interference (CRISPRi) system and a modular method to generate markerless mutants using CRISPR-Cas12a. Based on our findings, PCC 11901 is highly responsive to CRISPRi-based repression and showed high efficiencies for single insertion (31-81%) and multiplex double insertion (25%) genome editing with Cas12a. We envision that these tools will lay the foundations for the adoption of PCC 11901 as a robust model strain for engineering biology and green biotechnology

Lessons from the first generation of marine ecological forecast products

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Operation Moonshot: rapid translation of a SARS-CoV-2 targeted peptide immunoaffinity liquid chromatography-tandem mass spectrometry test from research into routine clinical use

OBJECTIVES: During 2020, the UK's Department of Health and Social Care (DHSC) established the Moonshot programme to fund various diagnostic approaches for the detection of SARS-CoV-2, the pathogen behind the COVID-19 pandemic. Mass spectrometry was one of the technologies proposed to increase testing capacity. METHODS: Moonshot funded a multi-phase development programme, bringing together experts from academia, industry and the NHS to develop a state-of-the-art targeted protein assay utilising enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) to capture and detect low levels of tryptic peptides derived from SARS-CoV-2 virus. The assay relies on detection of target peptides, ADETQALPQRK (ADE) and AYNVTQAFGR (AYN), derived from the nucleocapsid protein of SARS-CoV-2, measurement of which allowed the specific, sensitive, and robust detection of the virus from nasopharyngeal (NP) swabs. The diagnostic sensitivity and specificity of LC-MS/MS was compared with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) via a prospective study. RESULTS: Analysis of NP swabs (n=361) with a median RT-qPCR quantification cycle (Cq) of 27 (range 16.7-39.1) demonstrated diagnostic sensitivity of 92.4% (87.4-95.5), specificity of 97.4% (94.0-98.9) and near total concordance with RT-qPCR (Cohen's Kappa 0.90). Excluding Cq>32 samples, sensitivity was 97.9% (94.1-99.3), specificity 97.4% (94.0-98.9) and Cohen's Kappa 0.95. CONCLUSIONS: This unique collaboration between academia, industry and the NHS enabled development, translation, and validation of a SARS-CoV-2 method in NP swabs to be achieved in 5 months. This pilot provides a model and pipeline for future accelerated development and implementation of LC-MS/MS protein/peptide assays into the routine clinical laboratory

High-sensitivity troponin in the evaluation of patients with suspected acute coronary syndrome: a stepped-wedge, cluster-randomised controlled trial.

BACKGROUND: High-sensitivity cardiac troponin assays permit use of lower thresholds for the diagnosis of myocardial infarction, but whether this improves clinical outcomes is unknown. We aimed to determine whether the introduction of a high-sensitivity cardiac troponin I (hs-cTnI) assay with a sex-specific 99th centile diagnostic threshold would reduce subsequent myocardial infarction or cardiovascular death in patients with suspected acute coronary syndrome. METHODS: In this stepped-wedge, cluster-randomised controlled trial across ten secondary or tertiary care hospitals in Scotland, we evaluated the implementation of an hs-cTnI assay in consecutive patients who had been admitted to the hospitals' emergency departments with suspected acute coronary syndrome. Patients were eligible for inclusion if they presented with suspected acute coronary syndrome and had paired cardiac troponin measurements from the standard care and trial assays. During a validation phase of 6-12 months, results from the hs-cTnI assay were concealed from the attending clinician, and a contemporary cardiac troponin I (cTnI) assay was used to guide care. Hospitals were randomly allocated to early (n=5 hospitals) or late (n=5 hospitals) implementation, in which the high-sensitivity assay and sex-specific 99th centile diagnostic threshold was introduced immediately after the 6-month validation phase or was deferred for a further 6 months. Patients reclassified by the high-sensitivity assay were defined as those with an increased hs-cTnI concentration in whom cTnI concentrations were below the diagnostic threshold on the contemporary assay. The primary outcome was subsequent myocardial infarction or death from cardiovascular causes at 1 year after initial presentation. Outcomes were compared in patients reclassified by the high-sensitivity assay before and after its implementation by use of an adjusted generalised linear mixed model. This trial is registered with ClinicalTrials.gov, number NCT01852123. FINDINGS: Between June 10, 2013, and March 3, 2016, we enrolled 48 282 consecutive patients (61 [SD 17] years, 47% women) of whom 10 360 (21%) patients had cTnI concentrations greater than those of the 99th centile of the normal range of values, who were identified by the contemporary assay or the high-sensitivity assay. The high-sensitivity assay reclassified 1771 (17%) of 10 360 patients with myocardial injury or infarction who were not identified by the contemporary assay. In those reclassified, subsequent myocardial infarction or cardiovascular death within 1 year occurred in 105 (15%) of 720 patients in the validation phase and 131 (12%) of 1051 patients in the implementation phase (adjusted odds ratio for implementation vs validation phase 1·10, 95% CI 0·75 to 1·61; p=0·620). INTERPRETATION: Use of a high-sensitivity assay prompted reclassification of 1771 (17%) of 10 360 patients with myocardial injury or infarction, but was not associated with a lower subsequent incidence of myocardial infarction or cardiovascular death at 1 year. Our findings question whether the diagnostic threshold for myocardial infarction should be based on the 99th centile derived from a normal reference population. FUNDING: The British Heart Foundation

Characterising neutrophil subtypes in cancer using scRNA sequencing demonstrates the importance of IL-1β/CXCR2 axis in generation of metastasis specific neutrophils

Neutrophils are a highly heterogeneous cellular population. However, a thorough examination of the different transcriptional neutrophil states between health and malignancy has not been performed. We utilized single-cell RNA sequencing of human and murine datasets, both publicly available and independently generated, to identify neutrophil transcriptomic subtypes and developmental lineages in health and malignancy. Datasets of lung, breast, and colorectal cancer were integrated to establish and validate neutrophil gene signatures. Pseudotime analysis was used to identify genes driving neutrophil development from health to cancer. Finally, ligand–receptor interactions and signaling pathways between neutrophils and other immune cell populations in primary colorectal cancer and metastatic colorectal cancer were investigated. We define two main neutrophil subtypes in primary tumors: an activated subtype sharing the transcriptomic signatures of healthy neutrophils; and a tumor-specific subtype. This signature is conserved in murine and human cancer, across different tumor types. In colorectal cancer metastases, neutrophils are more heterogeneous, exhibiting additional transcriptomic subtypes. Pseudotime analysis implicates IL1β/CXCL8/CXCR2 axis in the progression of neutrophils from health to cancer and metastasis, with effects on T-cell effector function. Functional analysis of neutrophil-tumoroid cocultures and T-cell proliferation assays using orthotopic metastatic mouse models lacking Cxcr2 in neutrophils support our transcriptional analysis. We propose that the emergence of metastatic-specific neutrophil subtypes is driven by the IL1β/CXCL8/CXCR2 axis, with the evolution of different transcriptomic signals that impair T-cell function at the metastatic site. Thus, a better understanding of neutrophil transcriptomic programming could optimize immunotherapeutic interventions into early and late interventions, targeting different neutrophil states